️ 61: Monkeypox on the Frontline — Developing Brazil’s First qPCR Diagnostic Assay

️ Episode 61: Monkeypox on the Frontline — Developing Brazil’s First qPCR Diagnostic Assay In this episode of Base by Base, we trace the rapid development and rigorous validation of the first laboratory-developed qPCR test for monkeypox virus in Brazil’s Federal District. Back in July 2022, researchers at Sabin Diagnóstico e Saúde and the Catholic University of Brasília joined forces to design, optimize, and deploy a sensitive molecular assay that went from concept to clinical use in just 45 days. Along the way, they tackled challenges in sample collection, nucleic acid extraction, primer–probe design, and statistical modeling to ensure accurate, reliable detection of Mpox in patient specimens. Key highlights: the assay demonstrated 100 % accuracy and a limit of detection as low as 21.25 copies per reaction, showcasing exceptional sensitivity; careful optimization of input and elution volumes on both QiaSymphony and Maxwell platforms maximized viral recovery from pustule swabs; Probit regression on over 100 replicates underpinned a robust LoD estimation; targeted Sanger sequencing of four genomic regions confirmed perfect specificity and identity to reference sequences; a head-to-head comparison with the Federal District’s reference lab (LACEN-DF) yielded full concordance, securing official recognition for national surveillance reporting; and a retrospective epidemiological analysis of 295 tests revealed a 30 % overall positivity rate, pronounced male predominance, and spatial–temporal trends that illuminate the dynamics of Mpox spread in central Brazil. Conclusion: By integrating innovative extraction protocols, rigorous statistical validation, and collaborative cross-verification, this laboratory-developed test not only strengthened Brazil’s Mpox diagnostic capacity but also delivered crucial epidemiological insights, underscoring the power of agile molecular tools in outbreak response and public health preparedness. Reference: Silva, L.P.d.; Hurtado, F.A.; Belmok, A.; Correa, R.; Sousa, C.F.; Gil, G.P.; Velasco, L.; Jácomo, R.H.; Nery, L.F.; Rodrigues, M.T.d.O.; Andrade, M.S.; de Andrade, R.V. Development and Evaluation of a Molecular Test for Monkeypox Virus in the Federal District, Brazil. Genes 2025, 16, 779, https://doi.org/10.3390/genes16070779. License: This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International (CC BY 4.0) license – https://creativecommons.org/licenses/by/4.0/

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